The primary function of the skin is to serve as a protective barrier against the environment. Loss of the integrity of large portions of the skin as a result of wounds and wound-related infection may lead to major disability or even death. Every year in the United States more than 1.25 million people suffer burns and 6.5 million have chronic skin ulcers caused by diabetes and other diseases. As many as 15% of patients with diabetes will suffer from a diabetic foot ulcer and, of these patients, 6% will be hospitalized due to infection or other ulcer-related complications. Furthermore, diabetes is the leading cause of nontraumatic amputations of lower extremities in the United States, and approximately 14-24 % of patients with diabetes who develop a foot ulcer will have an amputation. Wounds in the elderly are also slower to heal and the problem is especially compounded in elderly diabetics. In addition, many traumatic wounds are incurred through accidents or from combat casualties and there is a need for an accelerated wound repair treatment for patients who undergo invasive surgery. Wounds incurred by members of the armed forces can be compounded by contamination and the risk of infection based on the environment in which the casualty is received. Consequently, wounds from traumatic injury, burns or diabetes pose an increased burden to the healthcare system and there is an immediate need for improved treatment options that promote and accelerate wound healing while minimizing or inhibiting the risk from infection during the healing process. Such a therapy which further promotes wound healing without scar formation would also be of significant benefit since scars over joints can limit limb motion and can have a strong psychological impact. Consequently, the primary goals in the treatment of wounds are rapid wound closure with minimal appearance of scarring. Recent advances in cellular and molecular biology have greatly expanded our understanding of the biologic processes involved in wound repair and tissue regeneration and have led to improvements in wound care. Cutaneous wound healing differs between fetal and adult skin. Wound repair in adult skin begins with an acute inflammatory phase and ends with the formation of a permanent scar. In contrast, early gestation fetal wounds heal in a near perfect fashion, rapidly and without the production of a scar. Amongst several other targets, factors such as transforming growth factor-ss1 (TGF-ss1), and COX-2 are upregulated in adult tissue and show reduced expression in fetal skin. Using RNA interference (RNAi) to down regulate a target gene expression, we have previously demonstrated that a multi-targeted siRNA cocktail targeting these genes can accelerate wound repair in acute wounds when administered in Histidine Lysine Polymer (HKP) - a branched cationic peptide. The treatment also showed reduced scarring and a return to normal histology of the skin. While these observations suggest a new therapeutic based on this approach, the topical administration of this material may not allow penetration across the tissue in the wound as reepithelialization occurs. The healing process itself may prevent further access of the siRNAs to the site of action within the wound bed. Chitosan - a cationic polysaccharide - has also been used in wound treatments and can also carry siRNAs. In this proposal Aim 1 seeks to examine whether chitosan and HKP can be modified with transdermal peptides while maintaining their siRNA carrying capability. Aim 2 will evaluate the best carrier from Aim 1 to see if it can increase the degree of penetration of the siRNA formulation through the skin. Upon identification of the optimal delivery vehicle (which may be unmodified), Aim 3 will evaluate the multi- targeted siRNA cocktail formulation in young versus elderly mice in animal models of wound healing. We will further examine the cocktail for efficacy in healing wounds in young and elderly diabetic mice. Demonstration of improved rate of wound repair in these models will be the first step towards migration of this therapy to the clinic to treat patients with similar conditions. Such a therapy may significantly improve wound healing in the elderly, reduce the complications from diabetes, burns or combat casualties and may help reduce the numbers of amputations that are performed on diabetic patients. PUBLIC HEALTH RELEVANCE: Ligand Directed Transdermal siRNA Delivery to Improve Wound Healing The primary function of the skin is to serve as a protective barrier against the environment. Loss of the integrity of large portions of the skin as a result of wounds and wound-related infection may lead to major disability or even death. Every year in the United States more than 1.25 million people suffer burns and 6.5 million have chronic skin ulcers caused by diabetes and other diseases. As many as 15% of patients with diabetes will suffer from a diabetic foot ulcer and, of these patients, 6% will be hospitalized due to infection or other ulcer-related complications. Furthermore, diabetes is the leading cause of nontraumatic amputations of lower extremities in the United States, and approximately 14-24 % of patients with diabetes who develop a foot ulcer will have an amputation. Wounds in the elderly are also slower to heal and the problem is especially compounded in elderly diabetics. In addition, many traumatic wounds are incurred through accidents or from combat casualties and there is a need for an accelerated wound repair treatment which diminishes scar formation in patients who undergo invasive or cosmetic surgery. Consequently, wounds in the elderly, from traumatic injury, burns or diabetes pose an increased burden to the healthcare system and there is an immediate need for improved treatment options that promote and accelerate wound healing while minimizing or inhibiting the risk from infection during the healing process. We have previously determined that delivery of two siRNAs silencing two gene targets in parallel to a wound (using a cationic peptide (HKP)) markedly improves the rate of wound closure and also reduced scarring in both mouse and pig excisional wound models. This project will seek to improve upon this observation by developing novel topical delivery vehicles that can further penetrate across the tissue within a wound. We will examine whether deeper siRNA delivery across a wound correlates with improved wound healing rate and further reduces scar formation in elderly animals and diabetic animal models. The outcome of this project will be the initial characterization of a valuable therapeutic option for treatment of patients with various types of skin wounds.